Defining the stoichiometry of inositol 1,4,5-trisphosphate binding required to initiate Ca2+ release (original) (raw)
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Journal of Biological Chemistry, 2013
Background: Proteolytic cleavage and disruption of inositol 1,4,5-trisphosphate receptor (IP 3 R) architecture may contribute to the initiation/progression of apoptosis. Results: Proteolytic cleavage products of the IP 3 R remain membrane-associated, and these fragments form functional tetrameric channels. Conclusion: Fragmentation of the IP 3 R does not inevitably lead to loss of function. Significance: Peptide continuity is not required for IP 3 R function, and IP 3 R activation may persist after protease cleavage during apoptosis. Inositol 1,4,5-trisphosphate receptor isoforms are a family of ubiquitously expressed ligand-gated channels encoded by three individual genes. The proteins are localized to membranes of intracellular Ca 2؉ stores and play pivotal roles in Ca 2؉ homeostasis. Previous studies have demonstrated that IP 3 R1 is cleaved by the intracellular proteases calpain and caspase both in vivo and in vitro. However, the resultant cleavage products are poorly defined, and the functional consequences of these proteolytic events are not fully understood. We demonstrate that IP 3 R1 is cleaved during staurosporine-induced apoptosis, yielding N-terminal fragments encompassing the ligand-binding domain and the majority of the central modulatory domain together with a C-terminal fragment containing the channel domain and cytosolic tail. Notably, these fragments remain associated with the membrane after initiation of apoptotic cleavage. Furthermore, when recombinant IP 3 R1 fragments, corresponding to those predicted to be generated by caspase or calpain cleavage, are stably coexpressed in cells, they physically associate and form functional channels. These data provide novel insights regarding the regulation of IP 3 R1 during proteolysis and provide direct evidence that polypeptide continuity is not required for IP 3 R activation and Ca 2؉ release.
Proceedings of the National Academy of Sciences, 2005
Virtually all functions of a cell are influenced by cytoplasmic [Ca 2؉ ] increases. Inositol 1,4,5-trisphosphate receptor (IP3R) channels, located in the endoplasmic reticulum (ER), release Ca 2؉ in response to binding of the second messenger, IP3. IP3Rs thus are part of the information chain interpreting external signals and transforming them into cytoplasmic Ca 2؉ transients. IP3Rs function as tetramers, each unit comprising an N-terminal ligand-binding domain (LBD) and a C-terminal channel domain linked by a long regulatory region. It is not yet understood how the binding of IP3 to the LBD regulates the gating properties of the channel. Here, we use the expression of IP3 binding protein domains tethered to the surface of the endoplasmic reticulum (ER) to show that the all-helical domain of the IP3R LBD is capable of depleting the ER Ca 2؉ pools by opening the endogenous IP3Rs, even without IP3 binding. This effect requires the domain to be within 50 Å of the ER membrane and is impaired by the presence of the N-terminal inhibitory segment on the LBD. These findings raise the possibility that the helical domain of the LBD functions as an effector module possibly interacting with the channel domain, thereby being part of the gating mechanisms by which the IP3-induced conformational change within the LBD regulates Ca 2؉ release.
Biochemical Journal, 1997
To elucidate the functional difference between type 1 and type 3 Ins(1,4,5)P3 receptors [Ins(1,4,5)P3R1 and Ins(1,4,5)P3R3 respectively] we studied the effect of Ca2+ on the ligand-binding properties of both Ins(1,4,5)P3R types. We expressed full-length human Ins(1,4,5)P3R1 and Ins(1,4,5)P3R3 from cDNA species in insect ovary Sf9 cells, and the membrane fractions were used for Ins(1,4,5)P3-binding assays. The binding of Ins(1,4,5)P3 to Ins(1,4,5)P3R1 and Ins(1,4,5)P3R3 was differentially regulated by Ca2+. With increasing concentrations of free Ca2+ ([Ca2+]), Ins(1,4,5)P3 binding to Ins(1,4,5)P3R1 decreased, whereas that to Ins(1,4,5)P3R3 increased. Alteration of Ins(1,4,5)P3 binding to Ins(1,4,5)P3R1 was observed at [Ca2+] ranging from less than 1 nM to more than 10 μM. The EC50 of Ins(1,4,5)P3 binding was 100 nM Ca2+ for Ins(1,4,5)P3R1. In contrast, Ins(1,4,5)P3 binding to Ins(1,4,5)P3R3 was changed at high [Ca2+] with an EC50 value of 872 nM, and steeply between 100 nM and 10 μM....
2007
The ubiquitous inositol 1,4,5-trisphosphate receptor (InsP 3 R) intracellular Ca 2 þ release channel is engaged by thousands of plasma membrane receptors to generate Ca 2 þ signals in all cells. Understanding how complex Ca 2 þ signals are generated has been hindered by a lack of information on the kinetic responses of the channel to its primary ligands, InsP 3 and Ca 2 þ , which activate and inhibit channel gating. Here, we describe the kinetic responses of single InsP 3 R channels in native endoplasmic reticulum membrane to rapid ligand concentration changes with millisecond resolution, using a new patch-clamp configuration. The kinetics of channel activation and deactivation showed novel Ca 2 þ regulation and unexpected ligand cooperativity. The kinetics of Ca 2 þ-mediated channel inhibition showed the singlechannel bases for fundamental Ca 2 þ release events and Ca 2 þ release refractory periods. These results provide new insights into the channel regulatory mechanisms that contribute to complex spatial and temporal features of intracellular Ca 2 þ signals.
Journal of Biological Chemistry, 1997
Structural and functional analyses were used to investigate the regulation of the inositol 1,4,5-trisphosphate (InsP 3 ) receptor (InsP 3 R) by Ca 2؉ . To define the structural determinants for Ca 2؉ binding, cDNAs encoding GST fusion proteins that covered the complete linear cytosolic sequence of the InsP 3 R-1 were expressed in bacteria. The fusion proteins were screened for Ca 2؉ and ruthenium red binding through the use of 45 Ca 2؉ and ruthenium red overlay procedures. Six new cytosolic Ca 2؉ -binding regions were detected on the InsP 3 R in addition to the one described earlier (Sienaert, I., De Smedt, H., Parys, J. B., Missiaen, L., Vanlingen, S., Sipma, H., and Casteels, R. (1996) J. Biol. Chem. 271, 27005-27012). Strong 45 Ca 2؉ and ruthenium red binding domains were localized in the N-terminal region of the InsP 3 R as follows: two Ca 2؉ -binding domains were located within the InsP 3 -binding domain, and three Ca 2؉ binding stretches were localized in a 500-amino acid region just downstream of the InsP 3 -binding domain. A sixth Ca 2؉ -binding stretch was detected in the proximity of the calmodulin-binding domain. Evidence for the involvement of multiple Ca 2؉ -binding sites in the regulation of the InsP 3 R was obtained from functional studies on permeabilized A7r5 cells, in which we characterized the effects of Ca 2؉ and Sr 2؉ on the EC 50 and cooperativity of the InsP 3 -induced Ca 2؉ release. The activation by cytosolic Ca 2؉ was due to a shift in EC 50 toward lower InsP 3 concentrations, and this effect was mimicked by Sr 2؉ . The inhibition by cytosolic Ca 2؉ was caused by a decrease in cooperativity and by a shift in EC 50 toward higher InsP 3 concentrations. The effect on the cooperativity occurred at lower Ca 2؉ concentrations than the inhibitory effect on the EC 50 . In addition, Sr 2؉ mimicked the effect of Ca 2؉ on the cooperativity but not the inhibitory effect on the EC 50 . The different [Ca 2؉ ] and [Sr 2؉ ] dependencies suggest that three different cytosolic interaction sites were involved. Luminal Ca 2؉ stimulated the release without affecting the Hill coefficient or the EC 50 , excluding the involvement of one of the cytosolic Ca 2؉ -binding sites. We conclude that multiple Ca 2؉ -binding sites are localized on the InsP 3 R-1 and that at least four different Ca 2؉ -interaction sites may be involved in the complex feedback regulation of the release by Ca 2؉ .
The Journal of General Physiology, 1998
The inositol (1,4,5)-trisphosphate receptor (InsP3R) mediates Ca2+release from intracellular stores in response to generation of second messenger InsP3. InsP3R was biochemically purified and cloned, and functional properties of native InsP3-gated Ca2+channels were extensively studied. However, further studies of InsP3R are obstructed by the lack of a convenient functional assay of expressed InsP3R activity. To establish a functional assay of recombinant InsP3R activity, transient heterologous expression of neuronal rat InsP3R cDNA (InsP3R-I, SI− SII+ splice variant) in HEK-293 cells was combined with the planar lipid bilayer reconstitution experiments. Recombinant InsP3R retained specific InsP3binding properties (Kd= 60 nM InsP3) and were specifically recognized by anti–InsP3R-I rabbit polyclonal antibody. Density of expressed InsP3R-I was at least 20-fold above endogenous InsP3R background and only 2–3-fold lower than InsP3R density in rat cerebellar microsomes. When incorporated i...
Molecular properties of inositol 1,4,5-trisphosphate receptors
Cell Calcium, 1999
The receptors for the second messenger inositol 1,4,5-trisphosphate (IP3) constitute a family of Ca2+ channels responsible for the mobilization of intracellular Ca2+ stores. Three different gene products (types I-III) have been isolated, encoding polypeptides which assemble as large tetrameric structures. Recent molecular studies have advanced our knowledge about the structure, regulation and function of IP3 receptors. For example, several Ca(2+)-binding sites and a Ca(2+)-calmodulin-binding domain have been mapped within the type I IP3 receptor, and studies on purified cerebellar IP3 receptors propose a second Ca(2+)-independent calmodulin-binding domain. In addition, minimal requirements for the binding of immunophilins and the formation of tetramers have been identified. Overexpression of IP3 receptors has provided further clues to the regulation of individual IP3 receptor isoforms present within cells, and the role that they play in the generation of IP3-dependent Ca2+ signals. Inhibition of IP3 receptor function and expression, and analysis of mutant IP3 receptors, suggests that IP3 receptors are involved in such diverse cellular processes as proliferation and apoptosis and are thus, necessary for normal development. Our understanding of the complex spatial and temporal nature of cytosolic Ca2+ increases and the role that these Ca2+ signals play in cell function depend upon our knowledge of the structure and the regulation of IP3 receptors. This review focuses on the molecular properties of these ubiquitous intracellular Ca2+ channels.
Journal of Biological Chemistry, 2013
Background: IP 3 R forms homo-and heterotetrameric channels. However, the impact of the specific composition of the heterotetrameric channel is undefined. Results: Concatenated IP 3 R dimers formed functional tetrameric channels. Heterotetrameric channels containing IP 3 R1 and IP 3 R2 had identical properties to IP 3 R2. Conclusion: Heterotetrameric IP 3 R do not behave as a blend of the constituent monomers. Significance: This study represents the first demonstration of the properties of heterotetrameric IP 3 R of unambiguously defined composition. Vertebrate genomes code for three subtypes of inositol 1,4,5trisphosphate (IP 3) receptors (IP 3 R1,-2, and-3). Individual IP 3 R monomers are assembled to form homo-and heterotetrameric channels that mediate Ca 2؉ release from intracellular stores. IP 3 R subtypes are regulated differentially by IP 3 , Ca 2؉ , ATP, and various other cellular factors and events. IP 3 R subtypes are seldom expressed in isolation in individual cell types, and cells often express different complements of IP 3 R subtypes. When multiple subtypes of IP 3 R are co-expressed, the subunit composition of channels cannot be specifically defined. Thus, how the subunit composition of heterotetrameric IP 3 R channels contributes to shaping the spatio-temporal properties of IP 3-mediated Ca 2؉ signals has been difficult to evaluate. To address this question, we created concatenated IP 3 R linked by short flexible linkers. Dimeric constructs were expressed in DT40-3KO cells, an IP 3 R null cell line. The dimeric proteins were localized to membranes, ran as intact dimeric proteins on SDS-PAGE, and migrated as an ϳ1100-kDa band on blue native gels exactly as wild type IP 3 R. Importantly, IP 3 R channels formed from concatenated dimers were fully functional as indicated by agonist-induced Ca 2؉ release. Using single channel "on-nucleus" patch clamp, the channels assembled from homodimers were essentially indistinguishable from those formed by the wild type receptor. However, the activity of channels formed from concatenated IP 3 R1 and IP 3 R2 heterodimers was dominated by IP 3 R2 in terms of the characteristics of regulation by ATP. These studies provide the first insight into the regulation of heterotetrameric IP 3 R of defined composition. Importantly, the results indicate that the properties of these channels are not simply a blend of those of the constituent IP 3 R monomers. Ca 2ϩ is an essential second messenger that controls a wide array of cell functions, including transcription, translation, secretion, contraction, memory, motility, fertilization, meiosis, mitosis, apoptosis, and autophagy (1-3). The variety of messages relayed by a change in [Ca 2ϩ ] necessitates that Ca 2ϩ signals are precise and unambiguous to ensure the activation of specific cellular processes with fidelity (4, 5). Each cell is equipped with a specific set of components of the "Ca 2ϩ toolkit," allowing an individual cell type to meet its particular physiological Ca 2ϩ signaling requirement (1, 6). Members of the IP 3 R 2 family constitute a major module of this signaling system (7). IP 3 R proteins are ligand-gated Ca 2ϩ channels localized in the membranes of intracellular Ca 2ϩ stores (8). IP 3 is formed in response to a variety of hormones, growth factors, or neurotransmitters that activate phospholipase C. It induces Ca 2ϩ mobilization from intracellular stores following binding to IP 3 R channels (9). Three subtypes of IP 3 R are encoded by different genes and are expressed in a tissue-and development-specific manner (10-13). Furthermore, multiple IP 3 R subtypes are often expressed in the same cell where they can form homomeric and heteromeric channels (14-18). Mammalian IP 3 R subtypes share 60-70% amino acid sequence identity and are organized with the same basic domain architecture (8, 19, 20). Each IP 3 R protein can be divided into an N-terminal ligand-binding domain, a central modulatory domain, and a C-terminal channel domain. Because of the high amino acid identity as well as structural conservation in the N and C termini, IP 3 R subtypes have the same single channel conductance and share similar IP 3-mediated gating properties (21-23). Nevertheless, the differences in primary sequence impart significantly different properties to the receptors; most notably, they differ in their affinities to various ligands and in their susceptibility to modulation by many * This work was supported, in whole or in part, by National Institutes of Health Grants DE14756 and DE19245. ࡗ This article was selected as a Paper of the Week.
Biochemical Society Transactions, 2015
Inositol 1,4,5-trisphosphate receptors (IP3Rs) are a family of ubiquitous, ER localized, tetrameric Ca2+ release channels. There are three subtypes of the IP3Rs (R1, R2, R3), encoded by three distinct genes, that share ∼60–70% sequence identity. The diversity of Ca2+ signals generated by IP3Rs is thought to be largely the result of differential tissue expression, intracellular localization and subtype-specific regulation of the three subtypes by various cellular factors, most significantly InsP3, Ca2+ and ATP. However, largely unexplored is the notion of additional signal diversity arising from the assembly of both homo and heterotetrameric InsP3Rs. In the present article, we review the biochemical and functional evidence supporting the existence of homo and heterotetrameric populations of InsP3Rs. In addition, we consider a strategy that utilizes genetically concatenated InsP3Rs to study the functional characteristics of heterotetramers with unequivocally defined composition. This ...